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eNeuro. 2017 Oct 4;4(5). pii: ENEURO.0181-17.2017. doi: 10.1523/ENEURO.0181-17.2017. eCollection 2017 Sep-Oct.

TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex.

Tomorsky J1,2, DeBlander L1,2, Kentros CG1,2,3,4, Doe CQ1,5,2,3, Niell CM1,2.

Author information

1
Institute of Neuroscience, University of Oregon, Eugene, OR 97403.
2
Department of Biology, University of Oregon, Eugene, OR 97403.
3
Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403.
4
Kavli Institute of Systems Neuroscience, NTNU, Trondheim 7030, Norway.
5
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

Abstract

Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA>GFPstop>HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the CA>GFPstop>HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.

KEYWORDS:

TU-tagging; gene expression; layer 2/3; postnatal; transcriptomics; visual cortex

PMID:
29085897
PMCID:
PMC5659240
DOI:
10.1523/ENEURO.0181-17.2017
[Indexed for MEDLINE]
Free PMC Article

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